**1. Introduction**

Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder affecting approximately 15–25% of the population, with higher prevalence in the female gender [1]. The pathophysiological mechanisms underlying IBS are not completely known. Different factors may participate in disease onset and perpetuation, including genetics, intestinal microbiota and low-grade inflammation [2]. In

addition, the role of the intestinal barrier in the pathogenesis of IBS is being increasingly recognised [3]. The loss of barrier integrity allows an increased passage of luminal antigens into the intestinal mucosa, hence stimulating the immune response and sensitizing the afferent nerve fibres [4].

Zonulin is a 47-kDa protein involved in the regulation of tight junctions (TJs), the primary determinants of paracellular permeability [5]. Serum zonulin is increased in various diseases in which alteration of intestinal permeability is central, including coeliac disease (CD), inflammatory bowel diseases (IBDs) and type 1 diabetes mellitus [6–8]. Moreover, higher serum zonulin levels were seen in patients with IBS compared to controls. Importantly, zonulin levels were directly correlated with the severity of bowel habits in patients with diarrhoea-predominant irritable bowel syndrome (IBS-D) [9].

Many studies have reported the presence of moderate immuno-inflammatory activity in both large and small intestine of patients with IBS [10]. Accordingly, patients with IBS showed increased levels of pro-inflammatory cytokines including interleukin (IL)-1b, tumour necrosis factor-alpha (TNFα), IL-6 and IL-8, and reduced levels of anti-inflammatory cytokines such as IL-10 [11], IL-5 and IL-13 [10]. Through the release of inflammatory cytokines such as TNFα and interferon-gamma (IFNγ), T lymphocytes contribute to TJ dysfunction [12,13].

Emerging literature data support a pathophysiological role of microbiome in IBS. The dysbiosis observed in subjects with IBS is characterized by reduced biodiversity, an increase in Bacteroides and Clostridia, and a reduction in Bifidobacteria [14,15]. There is also evidence of the ability of microbiome to regulate the intestinal barrier. The alteration of the commensal microbiota entails the presence of a morphologically aberrant intestinal mucosa, characterized by shorter ileal villi and smaller intestinal crypts, as demonstrated in germ-free mice [16]. In vitro studies showed that eubiotic microbiome favoured cell renewal process and expression of junctional proteins and mucins [17]. The reduced concentration in Bifidobacteria was associated with the severity of abdominal pain and number of bowel movements. Thus, targeting Bifidobacteria in the intestinal tract may alleviate microbiota-related diseases [18].

The rationale for the use of *Bifidobacterium longum* (*B. longum*) ES1 [19] in patients with IBS-D lies in its ability to modulate the microbiome improving intestinal dysbiosis [20], exert anti-inflammatory activity through the down-regulation of TNF-alpha and the up-regulation of IL-10 [21], and restore the integrity of the intestinal barrier, inducing the synthesis of TJ proteins [22,23].

Several meta-analyses have reported on the effectiveness and safety of probiotics in patients with IBS, especially for products containing Bifidobacteria and Lactobacilli. However, there was significant inter-study heterogeneity, warranting cautious interpretation of the findings. The heterogeneity mainly regarded differences in subgroup analyses of probiotics type, combination and dose, IBS subtype, symptomatic assessment scores and treatment duration [24,25]. Finally, not only probiotics but also prebiotics, such as inositol and beta-glucan, have been shown to improve symptoms in patients with IBS and in patients with concurrent IBD and IBS [26,27].

The aim of this pilot study was (1) to assess the use of *B. longum ES1* treatment in a homogeneous group of patients with IBS-D, by evaluating clinical response, through the use of validated questionnaires for IBS, and serological response, by the determination of serum levels of inflammatory cytokines and zonulin, in order to analyse potential effects on immune modulation and restoration of the intestinal barrier, respectively, and (2) to evaluate differences in response depending on the treatment duration. The results of this pilot study will allow us to define the optimal duration of probiotic therapy and to calculate the necessary sample size to conduct a subsequent randomized, double-blind, placebo-controlled clinical trial.

#### **2. Materials and Methods**
